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Veterinary microbiology2007; 123(1-3); 93-103; doi: 10.1016/j.vetmic.2007.02.004

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).

Abstract: A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.
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Publication Date: 2007-02-09 PubMed ID: 17346907DOI: 10.1016/j.vetmic.2007.02.004Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study presents a novel multiplex real-time PCR method developed to accurately detect and differentiate between two species of equid herpesviruses, EHV-1 and EHV-4. This method aids in the rapid diagnosis and differentiation of the two viruses in a single reaction, proving to be both sensitive and specific.

Study Methodology and Procedure

  • The researchers designed and developed a multiplex real-time PCR, targeting the glycoprotein B gene of EHV-1 and EHV-4. Specific primers and probes were used for each herpesvirus type, providing the facility to distinctively identify both viruses.
  • The study used two probes – minor-groove binding probes (MGB) with labels of 6-carboxy-fluorescein (FAM) and VIC, for the detection of EHV-1 and EHV-4 respectively.
  • A combination of EHV-1 and EHV-4 isolates, positive clinical samples, and reference strains were used in the study. These included ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain, three EHV-4 positive clinical samples, two EHV-4 isolates, and one EHV-4 reference strain.

Findings of the Study

  • The research showed that the EHV-1 isolates, positive clinical samples, and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR. A similar pattern was observed for the EHV-4 samples, indicating the ability of these tests to differentiate between EHV-1 and EHV-4.
  • There were no reactions observed when testing other herpesviruses, such as EHV-2, EHV-3, and EHV-5, demonstrating the specificity of the PCR method.
  • The multiplex real-time PCR didn’t show any reaction when tested with bacterial pathogens and opportunistic pathogens, reinforcing the specificity of this method.

Conclusions and Implications

  • Overall, the multiplex PCR showcases high sensitivity and specificity and serves as a valuable tool for the detection and differentiation of EHV-1 and EHV-4 in a single reaction.
  • The study effectively outlined an exhaustive equine herpesvirus disease investigation procedure, highlighting the importance of combining alphaherpesvirus multiplex real-time PCR with existing gel-based PCRs, providing a robust system for herpesvirus detection.

Cite This Article

APA
Diallo IS, Hewitson G, Wright LL, Kelly MA, Rodwell BJ, Corney BG. (2007). Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). Vet Microbiol, 123(1-3), 93-103. https://doi.org/10.1016/j.vetmic.2007.02.004

Publication

Veterinary microbiology
ISSN: 0378-1135
NlmUniqueID: 7705469
Country: Netherlands
Language: English
Volume: 123
Issue: 1-3
Pages: 93-103

Researcher Affiliations

Diallo, Ibrahim S
  • Animal Research Institute, Department of Primary Industries and Fisheries, Locked Mail Bag 4, Moorooka, Qld 4105, Australia. ibrahim.diallo@dpi.qld.gov.au
Hewitson, Glen
    Wright, Lucia L
      Kelly, Mark A
        Rodwell, Barry J
          Corney, Bruce G

            MeSH Terms

            • Animals
            • Herpesvirus 1, Equid / genetics
            • Herpesvirus 1, Equid / isolation & purification
            • Herpesvirus 4, Equid / genetics
            • Herpesvirus 4, Equid / isolation & purification
            • Polymerase Chain Reaction / methods
            • Polymerase Chain Reaction / veterinary
            • Rabbits
            • Sensitivity and Specificity

            Citations

            This article has been cited 23 times.
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